Date of Award
Doctor of Philosophy (PhD)
Annel K. Greene
Tzuen-Rong Jeremy Tzeng
Biological soil amendments of animal origin (BSAAO), such as animal waste or animal waste-based composts, commonly used as organic fertilizer, may contain human pathogens such as Salmonella and Listeria monocytogenes. To reduce harmful microorganisms, animal waste can be treated by composting or other validated scientific methods. But insufficient treatment may introduce pathogens into agricultural fields. As a nutrient-rich fertilizer, poultry litter may also contain human pathogens with Salmonella spp. as a primary focus. Physical heat treatments can kill Salmonella in poultry litter with or without the composting process, but validation studies or guidelines are still needed for the litter processing industry to ensure the microbial safety of their products. Further, due to the ubiquitous nature of L. monocytogenes, it is essential to understand the ecology of this pathogen where it inhabits and then develop strategies to reduce Listeria contamination. We hypothesized that compost-adapted competitive exclusion (CE) microorganisms against L. monocytogenes exist in animal waste-based compost. In combination with the culturing method, the use of next-generation sequencing approaches is expected to speed up the discovery of those compost-borne CE microorganisms for controlling L. monocytogenes in pre-harvest environments. Therefore, the objectives of this study were to 1) test a nonpathogenic surrogate microorganism for validating desiccation-adapted Salmonella inactivation in physically heat-treated broiler litter, 2) validate the physical heat treatment of poultry litter composts using surrogate and indicator microorganisms for Salmonella in industrial settings, 3) use next-generation sequencing approaches to understand the microbial community profile and functions in animal waste-based compost in the presence and absence of L. monocytogenes, and 4) isolate and identify the competitive exclusion microorganisms against L. monocytogenes in biological soil amendments.In order to test a non-pathogenic surrogate for validating desiccation-adapted Salmonella inactivation in physically heat-treated broiler litter, thermal resistance of desiccation-adapted S. ser. Senftenberg 775/W was compared with that of Enterococcus faecium NRRL B-2354 in aged broiler litter. Samples of aged broiler litter with 20, 30, and 40% moisture content were inoculated separately with desiccation-adapted S. Senftenberg 775/W and E. faecium NRRL B-2354 at ca. 5 to 6 log CFU/g, and then heat-treated at 75, 85, and 150°C. At all tested temperatures, desiccation-adapted E. faecium NRRL B-2354 was more heat-resistant than desiccation-adapted S. Senftenberg 775/W (P < 0.05). During the treatments at 75 and 85°C, E. faecium NRRL B-2354 in aged broiler litter with all moisture contents was reduced by 2.9- to 4.1-log, and was above the detection limit of direct plating (1.3 log CFU/g), whereas S. Senftenberg 775/W could not be detected by enrichment (> 5-log reductions) during holding time at these temperatures. At 150°C, E. faecium NRRL B-2354 in aged broiler litter with 20 and 30% moisture contents was still detectable by enrichment after heat exposure for up to 15 min, whereas S. Senftenberg 775/W in aged broiler litter with all moisture contents could not be detected throughout the entire treatment. Our results revealed that E. faecium NRRL B-2354 can be used as a surrogate for Salmonella to validate the thermal processing of poultry litter by providing a sufficient safety margin. This study provides a practical tool for poultry litter processors to evaluate the effectiveness of their thermal processing. Next, we used indicator and surrogate microorganisms for Salmonella to validate the processes for physically heat-treated poultry litter compost in both lab settings and commercial plants. Initial lab validation studies indicated that 1.2- to 2.7-log or more reductions of desiccation-adapted E. faecium NRRL B-2354 were equivalent to > 5-log reductions of desiccation-adapted Salmonella Senftenberg 775/W in poultry litter compost, depending on treatment conditions and compost types. Industrial plant validation studies were performed in one turkey litter processor and one laying hen litter processor. E. faecium was inoculated at ca.7-log CFU/g into the composted turkey litter and at ca. 5 log CFU/g into laying hen litter compost with respectively targeted moisture contents. The thermal processes in the two plants yielded reductions in E. faecium of 2.8 - > 6.4 log CFU/g (> 99.86%) of the inoculated. Similarly, for the processing control samples, reductions of presumptive indigenous enterococci were in the order of 1.8-3.7 log CFU/g (98.22% to 99.98 %) of the total naturally present. In contrast, there was less reduction of indigenous mesophiles (1.7-2.9 log CFU) and thermophiles (0.4-3.2 log CFU/g). Statistical analysis indicated that more indigenous enterococci were inactivated in the presence of higher moisture in the poultry litter compost. In conclusion, based on the data collected under the laboratory conditions, the processing conditions in both plants were adequate to reduce any potential Salmonella contamination of processed poultry litter material by at least 5-log, even though the processing conditions varied among trials and plants. Further, to understand the complex interactions between native compost microorganisms and L. monocytogenes, compost samples collected across the US were subjected to the inoculation of L. monocytogenes, and then systematically analyzed using 16S rRNA gene, shotgun-metagenomic, and metatranscriptomic sequencing approaches along with culturing methods. The reductions of L. monocytogenes in dairy and poultry compost with 40 or 80% moisture content at room temperature after 72 h of incubation ranged from 0.1 to 1.1 log CFU/g. Regrowth of L. monocytogenes occurred in some compost samples after 72 h of incubation, ranging from 0.1 to 1.5 log CFU/g. The major bacterial phyla identified in all farms are Firmicutes (23%), Proteobacteria (23%), Actinobacteria (19%), Chloroflexi (13%), Bacteroidetes (12%), Gemmatimonadetes (2%), and Acidobacteria (2%). The statistical analysis of sequencing data revealed that microbial interactions were affected by environmental factors such as compost types and location, moisture levels and incubation length, rather than the inoculation of L. monocytogenes. Although the similarities percentage (SIMPER) results are not significant for all samples, some specific genera (Bacillus, Sphaerobacter, Filomicrobium, Paucisalibacillus, Brumimicrobium, Steroidobacter Flavobacterium, or Chryseolinea) were identified as discriminant microorganisms contributing to the variation in community composition due to the presence of L. monocytogenes on multiple farms. After 72 h of incubation, changes in the metabolic pathways and the increased abundance of the bacteriocins category in the compost samples containing L. monocytogenes suggest that the interactions between L. monocytogenes and compost microbiome may include competition for compost nutrients and the presence of antimicrobials produced by the compost microbiome. Findings from this study clearly indicated that microbial diversity and functional profiles were significantly (P < 0.05) affected by the compost source, compost stage, and collection farm. Furthermore, the presence of specific discriminant microbial species may suggest certain compost samples as the potential sources for isolating CE microorganisms against L. monocytogenes. Competitive exclusion (CE) microorganisms have shown great potential as environmentally friendly tools to control harmful microorganisms. In consideration of dairy and poultry compost containing a diversity of microbial species, it was hypothesized that the compost may be a good source for isolating compost-borne CE microorganisms, which can inhibit the growth of Listeria monocytogenes. In this study, CE strains were screened and isolated from compost using double- or triple-agar-layer methods. The addition of resuscitation promoting factor (Rpf) produced by Micrococcus luteus promoted the growth of slow-growing/viable but non-culturable species from compost. A total of 40 bacterial isolates were confirmed with anti-L. monocytogenes activities, and then tested for Gram-reaction, motility, biofilm-forming ability, and inhibitory spectra against produce outbreak-associated L. monocytogenes and surrogate strains, followed by identification via 16S rRNA gene sequencing. About 50% of the isolated CE strains were identified as Bacillus spp., and 17 of 40 isolates can inhibit more than 10 produce outbreak-associated L. monocytogenes strains, while 9 CE strains isolated from poultry litter compost were confirmed as motile and competitive biofilm formers. Those 40 CE isolates based on the origin of isolation were separated into two groups, i.e. poultry and dairy CE groups, and then tested for anti-L. monocytogenes activity in both compost extracts and the compost. After 168 h incubation, the growth potentials of L. monocytogenes were reduced by co-culturing with CE strains in compost extracts under all conditions by 0.1- to 1.9- log depending on incubation temperature, types, and ratio of the compost extracts. Results showed that the inhibition effect from CE strains was higher in more concentrated compost extract (1:5) at 35°C or room temperature. In compost samples, the addition of CE strains can reduce L. monocytogenes population by ca. 1.2 log CFU/g at room temperature after 24 to 168 h incubation. The efficacy of CE strains was greater in the dairy compost as compared to that in the poultry litter compost. Findings from this study suggested that compost-adapted CE microorganisms have the potential as a biological control agent to control L. monocytogenes in agricultural environments. In summary, current processes for physically heat-treated poultry litter in industrial settings have been validated, In addition, this study provided tools (surrogate and/or indicator microorganism for Salmonella, and custom-designed sampler) for litter processors to modify their existing process parameters to produce Salmonella-free physically heat-treated poultry litter, which can be used by the produce industry to grow microbiologically safe products. Both compositional and functional changes in microbial communities of compost samples were studied, and the CE microorganisms with antagonistic activities against L. monocytogenes were identified. Based on metagenomics and culturing approaches, we have demonstrated that composts can be a rich source of CE microorganisms as potential biological control agents, which can be used for foodborne pathogen control in both preharvest and postharvest environments. Results generated from this study have provided both validation and biological control tools for ensuring microbiological safety of animal waste-based biological soil amendments.
Wang, Hongye, "Validation of Physically Heat-Treated Process for Poultry Litter and Analysis of Microbial Community of Animal Waste-Based Soil Amendments Using Sequencing Approach" (2020). All Dissertations. 2727.